Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner.

Sabine Nitschel,Tobias Nolden,Stefan Finke,Verena Te Kamp,Florian Pfaff,Kati Franzke,Luca M Zaeck,Dirk Höper,Madlin Potratz

doi:10.3390/v13101989

Abstract

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

Highlights

Rabies virus (RABV) and related lyssaviruses are highly neurotropic rhabdoviruses, which cause an almost invariably fatal disease once neurological symptoms appear [1]
The ectodomain of the RABV G protein is comprised of the fusion domain (FD), the pleckstrin homology domain (PHD), and the central domains (CD), which switch from a bent hairpin to an extended conformation by basic-to-acidic pH change [6]
Recombinant virus clones recombinant RABVs (rRABVs) Dog and rRABV Fox were derived from field virus cDNA clones prRABV Dog and prRABV Fox, and serially passaged on murine Na 42/13 neuroblastoma, fibroblastoid baby hamster kidney (BHK-21 clone BSR T7/5), and epithelial Madin–Darby canine kidney cells (MDCK-II) (Figure 1)

Summary

Introduction

Rabies virus (RABV) and related lyssaviruses are highly neurotropic rhabdoviruses, which cause an almost invariably fatal disease once neurological symptoms appear [1]. The negative sense ~12 kb RNA genome of RABV encodes for the five virus proteins: N (nucleoprotein), P (phosphoprotein), M (matrix protein), G (glycoprotein), and L (large polymerase). M and G are essentially involved in infectious virus particle release through budding at the plasma membrane [2]. The RABV glycoprotein G is located at the surface of RABV particles and is responsible for receptor binding and ribonucleoprotein (RNP) release in the cytoplasm by pHdependent membrane fusion. Essential for cell entry and in vivo spread [3], G is dispensable for virus particle release, resulting in the production of non-infectious particles in absence of G. For the production of infectious virus particles, G is an essential component and is simultaneously increasing the M-dependent budding efficiency [4,5]. The ectodomain of the RABV G protein is comprised of the fusion domain (FD), the pleckstrin homology domain (PHD), and the central domains (CD), which switch from a bent hairpin to an extended conformation by basic-to-acidic pH change [6]

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Conclusion